The C. Elegans Eggshell* Kathryn K
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The C. elegans eggshell* Kathryn K. Stein1,2 and Andy Golden1,§ 1Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA 2Translational Genomics Research Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA Table of Contents 1. Overview of the structure of the C. elegans eggshell ....................................................................... 2 2. Timeline for eggshell formation .................................................................................................. 3 3. Layer one: the vitelline layer ...................................................................................................... 4 4. Layer two: the chitin layer ......................................................................................................... 4 4.1. CHS-1 .........................................................................................................................5 4.2. GNA-2 ........................................................................................................................6 4.3. EGG-1, EGG-2 ............................................................................................................. 6 4.4. EGG-3, EGG-4, EGG-5 .................................................................................................. 6 4.5. SPE-11 ........................................................................................................................7 4.6. Other chitin-binding proteins ........................................................................................... 7 5. Layer three: the CPG layer ...................................................................................................... 17 5.1. Cortical Granules ........................................................................................................ 18 5.2. Components of the cortical granules ................................................................................ 20 6. Layer four: the extra-embryonic matrix ...................................................................................... 22 7. Layers five and six: the permeability barrier and the peri-embryonic layer ........................................ 23 8. Does actin play a role in eggshell formation? .............................................................................. 27 9. The block to polyspermy ......................................................................................................... 28 10. Screens for eggshell synthesis genes ........................................................................................ 30 11. Future ................................................................................................................................ 30 12. Table 1. Genes that when depleted cause the OID phenotype. ....................................................... 31 13. Acknowledgements .................................................................................................................................... 31 14. References .......................................................................................................................... 31 * Edited by Michel Labouesse and Donald G. Moerman Last revised February 20, 2015. Published December 30 2015. This chapter should be cited as: Stein K.K., Golden A. The C. elegans eggshell. (December 30, 2015), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.179.1, http://www.wormbook.org. Copyright: © 2015 Kathryn K. Stein and Andy Golden. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. §To whom correspondence should be addressed. E-mail: [email protected] 1 The C. elegans eggshell Abstract: In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern in which the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation. In all vertebrate and invertebrate species, oocytes are surrounded by an extracellular matrix (ECM) that is extensively modified upon fertilization. In each organism, the ECM serves similar purposes: (1) to mediate sperm binding, (2) to prevent the entry of supernumerary sperm, (3) to control the chemical environment of the embryo, and (4) to provide physical protection to the embryo as it encounters the environment in which it will develop. Although the function of the embryonic ECM is retained across species, it is not known whether the composition is conserved since the proteins that make up these structures are largely undefined. Generally, however, the ECM of many species is composed of glycoproteins. Also common to many organisms is a regulated exocytic event that is triggered after fertilization to build or modify the ECM (Wessel et al., 2001). In this chapter, we will describe the current understanding of the composition and synthesis of the C. elegans eggshell, highlighting the function of each layer and the genes known to be required for their assembly. 1. Overview of the structure of the C. elegans eggshell In the sections below, we will suggest some nomenclature changes based on conversations with those in the field and reviewers of this chapter. The first being that we number each layer according to the order in which they are deposited into the eggshell. The second suggestion is that we rename the spaces as layers as well. For many years, the C. elegans eggshell was known to be composed of three strata (Hall and Altun, 2008; Rappleye et al., 1999; Wharton, 1980; Bird, 1971) and the perivitelline space [now referred to as the extra-embryonic matrix (EEM), as suggested by a recent review (Johnston and Dennis, 2012)]. Two additional layers have recently been observed and defined (Olson et al., 2012; Benenati et al., 2009) (Figure 1). These layers are defined visually through electron micrographs and also by diagnostic biochemical treatments that can remove individual layers from the embryo (Olson et al., 2012; Benenati et al., 2009; Rappleye et al., 1999; Schierenberg and Junkersdorf, 1992). The outermost layer is the vitelline layer (VL) and can be selectively removed by treatment with sodium hypochlorite, leaving the remainder of the eggshell intact. This layer is present on the oocyte surface prior to fertilization. The next layer is the chitin layer (CL), digestible by chitinase. When this layer is removed, the embryo loses its ovoid shape and rounds up into a sphere. The third layer was previously thought to be a lipid-rich layer, based on chemical composition experiments done in Ascaris (Foor, 1967). However, Olson et al. (2012) have recently shown that this third layer is composed of proteoglycans. It is now referred to as the chondroitin proteoglycan (CPG) layer (Olson et al., 2012). The three outer layers are now known as the trilaminar outer eggshell. The fourth layer was previously known as the perivitelline space because it originally sat adjacent to the vitelline membrane, a name no longer used for the fifth layer (see Section 6 and Section 7). Johnston and Dennis (2012) suggested calling this layer the extra-embryonic matrix (EEM), a name we will use throughout this chapter. The EEM is an asymmetric layer that sits below the CPG layer (of the trilaminar outer eggshell). This layer is larger at the anterior end where the first extruded polar body resides. It is thought that this layer is fluid-filled and contains a number of proteins (Olson et al., 2012; Johnston et al., 2006). The fifth layer, which is separated from the trilaminar outer eggshell by the EEM, is now thought to be the lipid-rich layer, which makes up the osmotic/permeability barrier for the developing embryo. Olson et al. (2012) refer to this layer as the permeability barrier and its function is to keep out most small molecules and keep water in so the embryo stays hydrated. This layer has also been called the embryonic layer, due to its close apposition to the embryo (Benenati et al., 2009). Residing between the permeability barrier layer and the embryo proper is an amorphous space now called the peri-embryonic layer (layer six; Figure 1) (Olson et al., 2012). While Benenati et al. refer to the entire region as the embryonic layer, Olson et al. distinguish the permeability barrier as a thin membrane-like structure that encloses